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CN112920274A - Mouse monoclonal antibody for detecting alpha-synuclein protein and application thereof - Google Patents

Mouse monoclonal antibody for detecting alpha-synuclein protein and application thereof Download PDF

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Publication number
CN112920274A
CN112920274A CN202110285967.9A CN202110285967A CN112920274A CN 112920274 A CN112920274 A CN 112920274A CN 202110285967 A CN202110285967 A CN 202110285967A CN 112920274 A CN112920274 A CN 112920274A
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Prior art keywords
monoclonal antibody
alpha
synuclein protein
synuclein
protein
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CN202110285967.9A
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Inventor
李振韬
韩凤丽
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Jiangsu Begel Biomedical Co ltd
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Jiangsu Begel Biomedical Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The invention discloses a mouse monoclonal antibody for detecting alpha-synuclein protein, which can specifically identify the endogenous alpha-synuclein protein of cells, and also discloses a combined reagent, a kit or a test strip for detecting the alpha-synuclein protein. The invention has extremely high specificity to the endogenous alpha-synuclein protein of cells, and can be used as an important antibody reagent raw material for clinical research and screening.

Description

Mouse monoclonal antibody for detecting alpha-synuclein protein and application thereof
Technical Field
The invention discloses a mouse monoclonal antibody for detecting alpha-synuclein protein and application thereof, belonging to the field of immunology.
Background
Parkinson's Disease (PD) is a progressive and irreversible degenerative disease of the nervous system of dyskinesia, is the second most common primary neurodegenerative disease of the central nervous system, and patients mainly take middle-aged and elderly people as the main disease, cause a series of movement symptoms due to deficiency of dopamine neurotransmitter, and have the main clinical characteristics of resting tremor, muscular tension and stiffness, slow movement retardation and abnormal gait posture. The diagnosis of the Parkinson disease is mainly carried out according to clinical symptoms and physical signs of a patient and combined with the reactivity of the patient to dopaminergic disease, so that only middle and late stage cases can be found, and missed diagnosis and misdiagnosis are easy to occur. Therefore, in order to achieve early intervention and delay disease damage, the exploration and discovery of some biological indexes with high sensitivity and strong specificity for early diagnosis of PD becomes a problem to be solved urgently in clinical medicine.
alpha-synuclein (AS) is a precursor protein of non-beta amyloid, the gene is positioned at 4q21.3-q22 and consists of 6 exons and a plurality of introns, the coded AS consists of 140 amino acids, the N end contains 5-7 incomplete repetitive sequences (KTKEGV consensus motif) and is a facultative region of the protein; the central hydrophobic region (residues 61-95) contains a non-beta amyloid domain, which is an essential component for AS aggregation. Normally AS exists in a random coil form, in PD diseases, misfolded AS abnormally aggregates, deposits, forms lewy bodies (lewybodies), and through reducing dopamine release, increases metabolites, eventually leads to dopaminergic neuron degeneration, thereby affecting the occurrence of PD. Abnormal folding of AS plays a central role in the development of parkinson's disease and other synuclein aggregation disorders, and thus changes in AS are considered AS potential biomarkers that can respond to pathological changes in PD
There are some literature studies on the content change of AS in PD cases and body fluids of normal population, but the research results are very different, and the factor of the specificity of the AS antibody is not excluded. The patent provides a mouse monoclonal antibody capable of specifically recognizing cell endogenous AS protein, and the mouse monoclonal antibody has extremely high specificity.
Disclosure of Invention
In order to overcome the defects, the invention discloses a mouse monoclonal antibody and a kit for detecting alpha-synuclein protein.
The inventor finds and identifies a mouse monoclonal antibody for detecting alpha-synuclein protein, and the antibody is named as: 3F6, can specifically recognize the cell endogenous alpha-synuclein protein, and has extremely high specificity.
In more detail, the invention also provides the following technical scheme:
a mouse monoclonal antibody for detecting alpha-synuclein protein, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, the amino acid sequence of the light chain variable region is SEQ ID NO: 2.
furthermore, the combined reagent for detecting the alpha-synuclein protein comprises the murine monoclonal antibody.
Furthermore, the invention also provides a kit or a test strip, which comprises the mouse monoclonal antibody and other combined reagents.
The combined reagent, kit or test strip further comprises a color reagent for detecting an antibody, which is common in the prior art and is not described herein too much, and in addition, other additives, such as a buffer solution, a washing solution, etc., may be included in the kit, the components of the kit are provided in a predetermined ratio, and the relative amounts of the reagents are appropriately changed to maximize the sensitivity of the assay.
The invention has the beneficial effects that:
the invention provides a mouse monoclonal antibody capable of specifically recognizing cell endogenous AS protein, which has extremely high specificity and can be used AS an important antibody reagent raw material for clinical research and screening.
Drawings
FIG. 1 shows the monoclonal cell lines of 4 strains of the present invention which bind to the his-alpha-synuclein recombinant protein: 1B5, 3F6, 5A1 and 6H 5;
FIG. 2 shows SDS-PAGE electrophoresis of 4 monoclonal antibodies in the example of the present invention to determine purity;
FIG. 3 shows the results of ELISA detection of 4 monoclonal antibodies in the examples of the present invention;
FIG. 4 shows the immunochemical staining of the 4-strain monoclonal antibody of the present invention with SK-N-SH cells, HepG2 cells.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Example 1: preparation of anti-alpha-synuclein protein monoclonal antibody
Animal immunization
Three female BALB/C mice at 4-6 weeks are taken, his-alpha-synuclein recombinant protein is adopted as immunogen protein, complete Freund's adjuvant is emulsified and then injected subcutaneously at multiple points, and the immunization dose is 100 mu g/mouse per time. After three times of immunization, blood is collected by breaking the tail, the his-alpha-synuclein recombinant protein is used as a detection antigen (2 mu g/ml) to coat an enzyme label plate, the serum titer is determined by a gradient dilution method, and when the OD450 is 1.0, the serum titer is more than 1:4 w. One mouse with the best serum titer was selected for immunopotentiation, and the spleen was fused.
Cell fusion
Obtaining mouse spleen cells by a conventional method, fusing the mouse spleen cells with myeloma cells SP2/0 by a PEG method, wherein the number ratio of the fused cells is as follows: SP2/0 equals 1: 5. The fused cells were resuspended in HAT complete medium and plated in 96-well cell culture plates and cultured at 37 ℃ in a 5% CO2 incubator. When the hybridoma cells grew to a well area of 1/3 or more, culture supernatants were taken for ELISA detection. Meanwhile, the hybridoma cell wells are replaced by fresh HAT complete culture medium and are continuously cultured for 24h, and culture supernatant is taken for ELISA repeated detection.
Positive hybridoma cell selection
Screening was performed using indirect ELISA: using alpha-synuclein recombinant protein as a screening source, coating an enzyme label plate at the concentration of 2 mu g/ml, and coating overnight at 4 ℃; washing the plate washer PBST for three times, and sealing 5% skimmed milk powder at room temperature for 2 h; washing the plate washing machine PBST for three times, adding 100 mu l of hybridoma cell culture supernatant, and reacting for 1h at 37 ℃; washing the plate washing machine PBST for three times, adding a goat anti-mouse-HRP secondary antibody (Sigma A2554, 1:10000), and reacting for 1h at 37 ℃; washing the plate washer PBST for three times, adding 100 mu l of TMB, and developing for 10min at 37 ℃; the reaction was stopped by adding 50. mu.l of 2N H2SO 4; read at microplate reader OD450 nm. Test results were positive with OD450 readings greater than 1.0.
Monoclonal cell screening
And selecting positive cell wells, carefully blowing and beating the positive cell wells to prepare cell suspension, accurately calculating the concentration of the cell suspension according to a leukocyte counting method, and finally obtaining 10 hybridoma cells in each milliliter of HT complete medium through serial dilution. A96-well cell culture plate was prepared, and 100. mu.l of HT cell suspension was added to each well, i.e., each well theoretically contained only one cell, and cultured in a 5% CO2 incubator at 37 ℃. After 4 days the cell clusters (representing the number of single cells) were removed and observed, and the wells were labeled accordingly while changing 1/2 for fresh HT complete medium. After one week, the single cell culture supernatant was harvested for ELISA detection when the cells grew above the well area 1/3. And selecting cell holes with good cell growth state and strong positive value, transferring to 24 holes, culturing for 48h, and performing ELISA repeated detection. Test results were positive with OD450 readings greater than 1.5. If the positive single cell is not taken for the first time, the single cell screening process is repeated. 4 monoclonal cell strains of the his-alpha-synuclein recombinant protein are obtained through screening: 1B5, 3F6, 5A1 and 6H5 (shown in figure 1).
Preparation of monoclonal antibody
Female BALB/C mice of 8-10 weeks of age were selected and first pretreated by intraperitoneal injection of 0.5ml of liquid paraffin. The peritoneal cavity was inoculated with a suspension of monoclonal cells 1-2 weeks later in an amount of 1X107 cells/cell. After one week, the abdominal cavity of the mouse is observed and when the abdominal cavity of the mouse is obviously enlarged, the ascites is extracted by a syringe. 1ml of ascites fluid was diluted 4-fold with PBS and purified by Protein A/G affinity column chromatography. Collecting the effluent of protein peak, dialyzing with Phosphate Buffer Solution (PBS), measuring OD280 with ultraviolet spectrophotometer, calculating antibody protein concentration, detecting purity with SDS-PAGE electrophoresis, and detecting antibody titer with ELISA. The result of SDS-PAGE is shown in FIG. 2: the results show that all 4 monoclonal antibodies can be effectively purified, and the electrophoretic purity is more than 90%. The ELISA assay results are shown in FIG. 3: when His-alpha-synuclein recombinant protein is used as a detection antigen, the titer of 6H5 is the highest, and the titer of 5A1 is the lowest.
Example 2: screening of monoclonal antibody for specifically recognizing endogenous alpha-synuclein protein of cell
The cell lines used were: human neuroblastoma cells SK-N-SH expressing alpha-synuclein; and HepG2 cells from liver cancer tissues that do not express alpha-synuclein. Immunocytochemical staining experiments were performed on both cells. The specific experimental method is as follows:
SK-N-SH, HepG2 cells were inoculated into 96-well cell culture plates, respectively, and cultured at 37 ℃ for 24-36h with 5% CO 2; removing the culture medium, carefully rinsing with PBS twice, and fixing with 4% paraformaldehyde stationary liquid at room temperature for 15 min; rinsing with PBS for three times, each for 3 min; adding 100 μ l of 1% Triton X-100 into each well, standing at room temperature for 10 min; rinsing with PBS for three times, each for 3 min; adding 100 μ l of 3% hydrogen peroxide into each well, treating at room temperature for 10min, and blocking endogenous peroxidase; rinsing with PBS for three times, each for 3 min; adding purified anti-alpha-synuclein mouse monoclonal antibody, and incubating overnight at 4 ℃; PBST rinsing for four times, each time for 3 min; Anti-Mouse IgG (Fc specific) -peroxidase antibody (Sigma A2554) (1:1000) secondary antibody was added and incubated at 37 ℃ for 1 h; PBST rinsing for four times, each time for 3 min; adding DAB color development solution (ZLI-9017, China fir Jinqiao in Beijing), reacting at room temperature (reaction time is not more than 10min), observing color development result under microscope, and washing with distilled water to terminate color development.
The results are shown in FIG. 4:
the monoclonal antibody 3F6 has immunochemical staining reaction only with SK-N-SH cells expressing alpha-synuclein protein, but has no immunochemical staining reaction with HepG2 cells not expressing alpha-synuclein protein; 1B5 has immune staining reaction with SK-N-SH, and also has strong immune staining reaction with HepG 2; 5A1 and 6H5 had no immunostaining reaction with SK-N-SH and HepG 2. Namely, only 3F6 can specifically recognize the alpha-synuclein protein endogenous to the cells in the four monoclonal antibodies.
Example 3: monoclonal antibody gene calling
Total mRNA of the monoclonal cell line 3F6 was extracted with reference to the Invitrogen "TRIZOL Reagent" operating Manual. Reverse transcription was performed with reference to the Fermantas "RevertAid First Strand cDNA Synthesis Kit Specification, and 5 'and 3' end primers were designed and synthesized, and the obtained cDNA was used as a template to amplify a monoclonal antibody gene under reaction conditions: 94 ℃ for 1 min; 30s at 94 ℃; 57 ℃ for 30 s; 72 ℃, 1min (30 cycles); 72 ℃ for 5 min. The PCR product band was recovered, ligated to pMD-19T vector, and transformed into TOP10 competent cells, spread on LB plate with IPTG, X-gal, and subjected to blue-white screening. White single colonies were picked and inoculated into 2ml LB medium (Amp +), cultured overnight at 37 ℃ and the DNA of the extracted bacterial liquid was sequenced. The amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, the amino acid sequence of the light chain variable region is SEQ ID NO: 2.
the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Jiangsu Begel biopharmaceutical GmbH
<120> mouse monoclonal antibody for detecting alpha-synuclein protein and application thereof
<141> 2021-03-17
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Gly Val Ile Tyr Asp Gly Asp Gly Asp Thr Phe Tyr Ala Ser Trp Ala
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Lys Gly Arg Phe Thr Thr Ser Lys Thr Ser Ser Thr Thr Val Asp Leu
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Leu Ile Tyr Ser Tyr Thr Thr Leu Glu Ser Gly Val Pro Ser Arg Phe
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Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Gly Val
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Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Asn Ser Tyr
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Claims (3)

1. A mouse monoclonal antibody for detecting alpha-synuclein protein is characterized in that the mouse monoclonal antibody can specifically recognize the alpha-synuclein protein endogenous to cells, wherein the amino acid sequence of a heavy chain variable region of the mouse monoclonal antibody is SEQ ID NO: 1, the amino acid sequence of the light chain variable region of the murine monoclonal antibody is SEQ ID NO: 2.
2. a combined reagent for detecting alpha-synuclein protein, wherein the combined reagent comprises the murine monoclonal antibody of claim 1.
3. A kit or a test strip for detecting alpha-synuclein protein, comprising the murine monoclonal antibody of claim 1.
CN202110285967.9A 2021-03-17 2021-03-17 Mouse monoclonal antibody for detecting alpha-synuclein protein and application thereof Withdrawn CN112920274A (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102317316A (en) * 2008-12-19 2012-01-11 帕尼玛制药股份公司 Human anti-α-synuclein autoantibody
CN103796679A (en) * 2011-06-23 2014-05-14 比奥根艾迪克国际神经科学公司 Anti-alpha synuclein binding molecules
CN104215779A (en) * 2014-09-18 2014-12-17 首都医科大学宣武医院 Method for detecting combination of hemoglobin and alpha-synuclein
CN109475616A (en) * 2016-06-02 2019-03-15 麦迪穆有限责任公司 Antibodies against α -synuclein and uses thereof
CN110172098A (en) * 2019-05-27 2019-08-27 长春工业大学 The monoclonal antibody and its application of anti-alpha-synapse nucleoprotein
CN110506057A (en) * 2017-02-17 2019-11-26 百时美施贵宝公司 ALPHA synapse nucleoprotein antibody and its application

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102317316A (en) * 2008-12-19 2012-01-11 帕尼玛制药股份公司 Human anti-α-synuclein autoantibody
CN103796679A (en) * 2011-06-23 2014-05-14 比奥根艾迪克国际神经科学公司 Anti-alpha synuclein binding molecules
CN104215779A (en) * 2014-09-18 2014-12-17 首都医科大学宣武医院 Method for detecting combination of hemoglobin and alpha-synuclein
CN109475616A (en) * 2016-06-02 2019-03-15 麦迪穆有限责任公司 Antibodies against α -synuclein and uses thereof
CN110506057A (en) * 2017-02-17 2019-11-26 百时美施贵宝公司 ALPHA synapse nucleoprotein antibody and its application
CN110172098A (en) * 2019-05-27 2019-08-27 长春工业大学 The monoclonal antibody and its application of anti-alpha-synapse nucleoprotein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
李尧华等: "揭示α-突触核蛋白与帕金森病的关系：α-突触核蛋白单克隆抗体的研究", 《中国临床康复》 *

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Application publication date: 20210608